In recent years, amounts of trace protein and the like contained in biological samples, such as sera, urine and the like, can be measured by an enzyme immunoassay, such as so-called sandwich immunoassay, competitive immunoassay and the like, using antibodies and antigens. For example, in the sandwich immunoassay, a sandwich complex (immobilized antibody--antigen--labeled antibody) in correlation with the amount of an antigen to be measured (referred to as an "analyte") is formed from an immobilized monoclonal antibody (immobilized antibody) specific for the analyte and an enzyme-linked monoclonal antibody which is different from the above monoclonal antibody but specific for the analyte (labeled antibody), a labeled antibody remained after the sandwich complex formation is separated, and then the amount of the enzyme in the sandwich complex is measured based on its activity to finally estimate the amount of the analyte.
Enzyme substrates for use in respective enzyme reactions are provided generally in the form of solution or freeze-dried powder. When an enzyme reaction is carried out, the substrate solution is used directly or after dilution or the freeze-dried powder is used after making it into a solution by adding water or the like. However, when the enzyme substrate is deteriorated, it causes bad influences upon the results of enzyme activity measurement, such as poor reproducibility and the like. In consequence, for manufacturers who produce and provide enzyme substrates, it is necessary to provide enzyme substrates which are stable for a prolonged period of time so that their reactivity with enzymes does not change.
In the enzyme immunoassay described above, enzymes are used as labels for measuring analytes. In addition, other techniques are frequently used such as a method in which an enzyme amount in a body fluid is determined by measuring its enzyme activity.
For example, JP-A-2-188578 (the term "JP-A" as used herein means an "unexamined published Japanese patent application") discloses that enzyme substrates are stabilized under alkaline conditions. However, since each enzyme has its own optimum pH range for generating its activity, the use of an enzyme substrate stabilized under alkaline conditions in this manner directly in the measurement of enzyme activity will reduce the enzyme activity itself when the alkaline pH does not coincide with the optimum pH of the enzyme to be measured, thereby causing a problem of decreased measuring accuracy. It is possible as a matter of course to adjust the pH value of a substrate solution stabilized under alkaline conditions to the optimum pH of the target enzyme, but it requires an additional step for adjusting a pH value prior to the measurement of enzyme activity.
In addition, U.S. Pat. No. 5,143,825 discloses a method for stabilizing an enzyme solution, which comprises adding a metal chelating agent, such as EDTA, EGTA or the like, to an enzyme substrate solution. In this method, 4-methylumbelliferone phosphoric acid or a salt thereof is stabilized by capturing metal ions which non-enzymatically hydrolyze the substrate. However, when 4-methylumbelliferone phosphoric acid or a salt thereof is used in this method as the substrate of alkaline phosphatase or the like which is frequently used in immunoassays but requires magnesium and zinc ions for generating its activity, these ions are also captured by the chelating agent to inhibit the enzyme activity, so that it is necessary to carry out an additional step such as removal of the chelating agent prior to use of the substrate solution.
In this connection, it is possible to improve stability of an enzyme substrate by freeze-drying or the like as compared with storage as a solution. However, even in the freeze-drying treatment, it would be convenient for users if an enzyme substrate capable of maintaining its quality more stably for more prolonged period of time could be provided. That is, this is because, since the freeze-dried enzyme substrate is dissolved in an appropriate solution prior to use, a large amount of stock solution can be prepared and stored by improving the storage stability of the substrate after dissolving even if the substrate is freeze-dried.